Normally, UV/Vis photometers in HPLC are predominantly employed in the so-called analytical scale in conjunction with a separation column having an internal diameter between 4.6 and 3 mm. In the case of columns with a smaller internal diameter, other, special flow cells are used. These cells have smaller volumes to minimize mixing and peak propagation.
If the column diameters become smaller, then the peak volumes are likewise smaller. To cleanly separate the peaks from one another, flow cells, with corresponding smaller volumes, are needed. As a result, the illuminated volume or, in other words, the volume through which light shines, and the diameter are likewise smaller. The transmission of the cell decreases, and, proportionately to this, the signal noise of the photodiodes increases.
In detection, the signal-to-noise ratio is critical. Hence the limit of detection (LOD) is located at approximately a minimum signal-to-noise (S/N) ratio of 3:1. In other words, a signal can be distinguished from the noise only whenever the ratio is at least three to one.
For a reliable limit of quantification (LOQ), an S/N ratio of at least 10:1 to 20:1 should be achieved. Some regulations even prescribe a ratio of 30:1.
At present, in cell and protein research, devices are needed that can operate with the requisite reduction in the quantity of substance. Since the substance quantity is at femtomole limits and even less, columns with a diameter of 75 μm (micrometers) or even smaller are used. In this case, only flow cells with a volume of 3 to 5 nl (nanoliters) are used.
Such cells generally comprise a quartz capillary with an internal diameter of 20 to 25 μm and an outside diameter of 280 μm. In other words, a gap of only 300 μm including the capillary course is available to the light on the way to the photodiode.
Attempts are made to compensate for this low light transmission by focusing the light, both on entering and on exiting the cell, by means of lenses. Although this does increase the quantity of light and reduces noise, the signal-to-noise ratio is nevertheless still unsatisfactory.
Commercial detectors on the market attempt to make the best of the situation by means of their internal firmware. The light coming from the optical grating is split into a reference channel and a sample channel, with the ratio of the split being invariable because of the hardware construction. The effective exposure times of the reference and sample photodiodes are usually regulated automatically such that the reference diode signal remains securely below the saturation limit. In turn, for the sample diode, this setting is inadequate for appropriately optimizing and reducing the resultant noise.
Examples of photometric devices with a flow cell are known from EP 0071645 B1, DE 695 24 405 T2, and DE 4308202 A1.